Acellular tissue matrices made from alpha-1,3-galactose-deficient tissue

ABSTRACT

The invention provides acellular tissue matrices made from collagen-containing tissues of animals genetically modified so as to be deficient in the galactose 1,3-galactose epitope and methods of making and using such acellular tissue matrices.

This application is a continuation of U.S. application Ser. No.10/896,594, filed Jul. 21, 2004, now abandoned which claims the benefitof U.S. Provisional Application No. 60/489,245, filed Jul. 21, 2003, thedisclosure of which is incorporated herein by reference in its entiretyall of which are incorporated herein by reference.

TECHNICAL FIELD

This invention relates to tissue matrices for tissue repair, and moreparticularly to acellular tissue matrices made from animals geneticallymodified so that their tissues are deficient in the galactose α-1,3galactose epitope.

BACKGROUND

A major problem of xenotransplantation in recipient animals (e.g.,humans) that do not express the enzyme UDP-galactose:β-D-galactosyl-1,4-N-acetyl-D-glucosaminide α-1,3 galactosyl-transferase(α-1,3 galactosyltransferase; “αGT”) that catalyzes the formation of theterminal disaccharide structure, galactose α-1,3 galactose (“αgal”), isthe hyperacute rejection of xenografts in such recipients that islargely, if not exclusively, due to the action of antibodies specificfor the αgal epitope on the surface of cells in the xenograft.Transgenic animals (e.g., pigs) have been derived which lack, orsubstantially lack, functional αGT and thus also lack, or substantiallylack, αgal epitopes.

SUMMARY

The invention is based in part on the discovery that acellular dermalmatrices (ADM) made from transgenic pigs in which both alleles of thegene encoding αGT have been disrupted such that the pigs lack thepredominant αGT activity (and thus αgal epitopes on the surface of cellsand on proteins of the extracellular matrix) did not stimulate theproduction of αgal-specific antibodies and were not infiltrated byinflammatory cells subsequent to implantation in Old World primates. Theinvention thus provides acellular tissue matrices made from animals(e.g., pigs) genetically modified so as to lack, or substantially lack,αgal epitopes and methods of making and using such matrices. Appropriategenetically modified animals will preferably be αGT gene-disruptedanimals, i.e., animals in which both alleles of the αGT gene have beendisrupted in all cells of the animal. For convenience, αGTgene-disrupted animals are referred to herein as “GTδ” animals andanimals that naturally lack functional αGT protein are referred to as“GT−” animals. Animals that naturally express functional αGT protein aresometimes referred to as “GT+” animals.

More specifically, the invention features an acellular tissue matrixthat includes: a decellularized collagen-containing tissue, or adecellularized collagen-containing organ, of an animal geneticallymodified such that tissues in the animal lack, or substantially lack,galactose α-1,3-galactose epitopes. The acellular tissue matrixpreferably lacks, or substantially lacks, epithelial basement membrane.The animal can be, for example, a pig. The tissue or organ can include,for example, dermis, fascia, pericardium, dura, umbilical cord,placenta, cardiac valve, ligament, tendon, artery, vein, neuralconnective tissue, intestine, bladder, or ureter. The acellular tissuematrix can be in non-particulate or in particulate form. The geneticmodification can include a disruption of both alleles of an α-1,3galactosyl transferase (αGT) gene.

Also embodied by the invention is a method of making an acellular tissuematrix. The method includes: (a) providing a collagen-containing tissueor a collagen-containing organ from an animal genetically modified sothat tissues in the animal lack, or substantially lack, galactoseα-1,3-galactose epitopes; and (b) processing the tissue or organ to soas to render the tissue or organ acellular and lacking, or substantiallylacking, in epithelial basement membrane, the processing resulting inthe production of an acellular tissue matrix. The animal can be a pigand the tissue or organ can any of the above-recited tissues or organs.The genetic modification can include a disruption of both alleles of anα-1,3 galactosyl transferase (αGT) gene. The method can further includefreezing and/or freeze-drying the acellular tissue matrix. Moreover, themethod can further include: (a) pulverizing the acellular tissue matrix;or (b) rendering the acellular tissue matrix particulate in form. Theprocessing can include removing and discarding an epithelium (e.g.,epidermis).

Another aspect of the invention is a method of treatment. The methodincludes: (a) identifying a mammalian subject as having an organ, ortissue, in need of repair or amelioration; and (b) placing a compositioncomprising the above-described acellular tissue matrix in or on theorgan or tissue. The subject can be, e.g., a human and the animal canbe, e.g., a pig. The tissue or organ can be any of those recited aboveand the acellular tissue matrix can be non-particulate or particulate inform. The genetic modification can include disruption of both alleles ofan α-1,3 galactosyl transferase (αGT) gene. The method can furthercomprise administration to the subject of one or more agents, e.g., acell growth factor, an angiogenic factor, a differentiation factor, acytokine, a hormone, or a chemokine. The one or more agents can be inthe composition placed in the subject or they can be injected or infusedinto the subject separately from the composition. The organ or tissue ofthe subject can be, without limitation, skin, bone, cartilage, meniscus,dermis, myocardium, periosteum, artery, vein, stomach, small intestine,large intestine, diaphragm, tendon, ligament, neural tissue, striatedmuscle, smooth muscle, bladder, urethra, ureter, gingival, or fascia(e.g., abdominal wall fascia). The composition can further includedemineralized bone powder. The gingiva can be, or can be proximal to,receding gingival. Moreover, the gingiva can contain a dental extractionsocket.

As used herein, the term “placing” a composition includes, withoutlimitation, setting, injecting, infusing, pouring, packing, layering,spraying, and encasing the composition. In addition, placing “on” arecipient tissue or organ means placing in a touching relationship withthe recipient tissue or organ.

As used herein, a “genetically modified” animal is an animal whosegenome contains an artificially inserted exogenous nucleic acid sequenceor whose genome is artificially manipulated so as to lack a wild-typenucleic acid sequence. Thus, a genetically modified animal is not one inwhich an exogenous sequence in its genome or the absence of a wild-typenucleic acid sequence from its genome is derived by only a breedingprogram as is used in, for example, the generation of congenic animalstrains. In addition, a genetically modified animal is not one intowhose genome a wild-type viral nucleic acid sequence has integrated inthe course of a viral infection. Genetically modified animals includethe progeny of the manipulated animal who carry the modification intheir genomes.

As used herein, the tissues in “an animal that is genetically modifiedsuch that the tissues in the animal substantially lack αgal epitopes”contain less than 5% (e.g., less than: 4%; 2%; 1%; 0.1%; 0.01%; 0.001%;or even less than 0.001%) of the αgal epitopes that correspondingtissues in a corresponding wild-type animal contain.

As used herein, the term “operably linked” means incorporated into agenetic construct so that expression control sequences (i.e.,transcriptional and translational regulatory elements) effectivelycontrol expression of a coding sequence of interest. Transcriptional andtranslational regulatory elements include but are not limited toinducible and non-inducible promoters, enhancers, operators and otherelements that are known to those skilled in the art and that drive orotherwise regulate gene expression. Such regulatory elements include butare not limited to the cytomegalovirus hCMV immediate early gene, theearly or late promoters of SV40 adenovirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of phage A, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase, the promoters of acid phosphatase, andthe promoters of the yeast α-mating factors.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In case of conflict, thepresent document, including definitions, will control. Preferred methodsand materials are described below, although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention. All publications, patentapplications, patents and other references mentioned herein areincorporated by reference in their entirety. The materials, methods, andexamples disclosed herein are illustrative only and not intended to belimiting.

Other features and advantages of the invention, e.g., acellular tissuematrices that are useful for implantation in GT− subjects (e.g., humanpatients), will be apparent from the following description, from thedrawings and from the claims.

DESCRIPTION OF DRAWINGS

FIGS. 1A-1D are series of photographs showing the staining AlloDerm(FIG. 1A), XenoDerm (FIG. 1B) and GalDerm (FIG. 1C) by a monoclonalantibody specific for the αgal epitope and horseradishperoxidase-conjugated secondary antibody. FIG. 1D shows the staining ofGalDerm exposed to the secondary antibody and not to the αgal-specificmonoclonal antibody.

FIGS. 2A and B are photographs of a hematoxylin and eosin (H&E) stainedAlloDerm sample removed from an African green monkey (Old World primate)35 days after implantation in the monkey. Photographs were taken at 40×(FIG. 2A) and 100× (FIG. 2B) magnification. The field shown in FIG. 2Bis indicated by a rectangle in FIG. 2A.

FIGS. 3A-C are photographs of a H&E stained XenoDerm sample removed froman African Green monkey (Old World primate) 35 days after implantationin the monkey. Photographs were taken at 40× (FIG. 3A), 100× (FIG. 3B),and (FIG. 3C) 400× magnification. The field shown in FIG. 3B isindicated by a rectangle in FIG. 3A and the field shown in FIG. 3C isindicated by a rectangle in FIG. 3B.

FIGS. 4A and B are photographs of a H&E stained GalDerm sample removedfrom an African Green monkey (Old World primate) 35 days afterimplantation in the monkey. Photographs were taken at 40× (FIG. 4A) and100× (FIG. 4B) magnification. The field shown in FIG. 4B is indicated bya rectangle in FIG. 4A.

FIGS. 5A-C are a series of line graphs showing the titers ofimplant-specific IgG antibodies (as measured by ELISA) in the sera ofindividual monkeys (3 per group) at 0, 7, 21, and 35 days afterimplantation of XenoDerm™ (FIG. 5A), GalDerm™ (FIG. 5B), or AlloDerm®(FIG. 5C). OD₄₀₅ values are shown on the Y-axes and reciprocal values ofserum dilutions (logarithmic scale) in the ELISA (enzyme-linkedimmunosorbent assay) are shown on the X-axes. Individualmonkey-identifying numbers are indicated above each graph.

DETAILED DESCRIPTION

The experiments described in Examples 1-4 indicate that: (a) acellulartissue matrices produced from GTδ animals do not stimulate theproduction of antibodies specific for the acellular tissue matrices inGT− recipient animals into which the acellular tissue matrices have beenimplanted; and (b) antibodies in such GT− recipient animals do notresult in an inflammatory reactions in implanted acellular tissuematrices from GTδ animals.

Various aspects of the invention are described below.

Acellular Tissue Matrices

As used herein, an “acellular tissue matrix” is a matrix that: (a) ismade from any of a wide range of collagen-containing tissues; (b) isacellular (i.e., is free of intact cells, alive or dead cells); and (c)retains many of the biological and structural functions possessed by thenative tissue or organ from which it was made. In addition, theacellular tissue matrices of the invention lack, or substantially lack,an epithelial basement membrane. The epithelial basement membrane is athin sheet of extracellular material contiguous with the basilar aspectof epithelial cells. Sheets of aggregated epithelial cells form anepithelium. Thus, for example, the epithelium of skin is called theepidermis and the skin epithelial basement membrane lies between theepidermis and the dermis. The epithelial basement membrane is aspecialized extracellular matrix that provides a barrier function and anattachment surface for epithelial-like cells; however, it does notcontribute any significant structural or biomechanical role to theunderlying tissue (e.g., dermis). Unique components of epithelialbasement membranes include, for example, laminin, collagen type VII, andnidogen. The unique temporal and spatial organization of the epithelialbasement membrane distinguish it from, e.g., the dermal extracellularmatrix. The presence of the epithelial basement membrane in an acellulartissue matrix of the invention could be disadvantageous in that theepithelial basement membrane likely contains a variety ofspecies-specific components that would elicit the production ofantibodies, and/or bind to preformed antibodies, in xenogeneic graftrecipients of the acellular matrix. In addition, the epithelial basementmembrane can act as barrier to diffusion of cells and/or soluble factors(e.g., chemoattractants) and to cell infiltration. Its presence inacellular tissue matrix grafts can thus significantly delay formation ofnew tissue from the acellular tissue matrix in a recipient animal. Asused herein, an acellular tissue matrix that “substantially lacks” anepithelial basement membrane is an acellular tissue matrix containingless than 5% (e.g., less than: 3%; 2%; 1%; 0.5%; 0.25%; 0.1%; 0.01%;0.001%; or even less than 0.001%) of the epithelial basement membranepossessed by the corresponding unprocessed tissue from which theacellular tissue matrix was derived.

Biological functions retained by the acellular tissue matrices of theinvention include cell recognition and cell binding as well as theability to support cell spreading, cell proliferation, and celldifferentiation. Such functions are provided by undenatured collagenousproteins (e.g., type I collagen) and a variety of non-collagenousmolecules (e.g., proteins that serve as ligands for other molecules suchas integrin receptors, molecules with high charge density such asglycosaminoglycans (e.g., hyaluronan) or proteoglycans, or otheradhesins). Structural functions retained by useful acellular tissuematrices include maintenance of histological architecture, maintenanceof the three-dimensional array of the tissue's components and physicalcharacteristics such as strength, elasticity, and durability, definedporosity, and retention of macromolecules. The efficiency of thebiological functions of an acellular tissue matrix can be measured, forexample, by its ability to support cell proliferation and is at least50% (e.g., at least: 50%; 60%; 70%; 80%; 90%; 95%; 98%; 99%; 99.5%;100%; or more than 100%) of those of the native tissue or organ fromwhich the acellular tissue matrix is made.

It is not necessary that the grafted matrix material be made from tissuethat is identical to the surrounding recipient tissue but should simplybe amenable to being remodeled by invading or infiltrating cells such asdifferentiated cells of the relevant host tissue, stem cells such asmesenchymal stem cells, or progenitor cells. Remodeling is directed bythe above-described acellular tissue matrix components and signals fromthe surrounding host tissue (such as cytokines, extracellular matrixcomponents, biomechanical stimuli, and bioelectrical stimuli). Thepresence of mesenchymal stem cells in the bone marrow and the peripheralcirculation has been documented in the literature and shown toregenerate a variety of musculoskeletal tissues [Caplan (1991) J.Orthop. Res. 9:641-650; Caplan (1994) Clin. Plast. Surg. 21:429-435; andCaplan et al. (1997) Clin Orthop. 342:254-269]. Additionally, the graftmust provide some degree (greater than threshold) of tensile andbiomechanical strength during the remodeling process.

It is understood that an acellular tissue matrix of the invention can beproduced from any collagen-containing tissue (e.g., dermis, fascia,pericardium, dura, umbilical cords, placentae, cardiac valves,ligaments, tendons, cartilage, bone, vascular tissue (arteries and veinssuch as saphenous veins), neural connective tissue, or ureters), as longas the above-described properties are retained by the matrix. Moreoverthe tissues in which the acellular tissue matrix grafts are placedinclude essentially any tissue that can be modeled by invading orinfiltrating cells (see above). Relevant tissues include skeletaltissues such as bone, cartilage, ligaments, fascia, and tendon. Othertissues in which any of the above acellular tissue matrix grafts can beplaced include, without limitation, fascia, skin, gingiva, dura,myocardium, vascular tissue, neural tissue, striated muscle, smoothmuscle, bladder wall, ureter tissue, intestine, and urethra tissue. Itis understood that, for the purposes of the invention, heart muscle andskeletal muscle are not the same tissue. See, U.S. Patent ApplicationPublication No. US2004/0043006 (the disclosure of which is incorporatedherein by reference in its entirety) for a description of epithelia,epithelial basement membranes, and components on the side of theepithelial basement membrane opposite to the epithelium in tissues suchas urinary bladder tissue and small intestine.

The form in which the acellular tissue matrix is provided will depend onthe tissue or organ from which it is derived and on the nature of therecipient tissue or organ, as well as the nature of the damage or defectin the recipient tissue or organ. Thus, for example, a matrix derivedfrom a heart valve can be provided as a whole valve, as small sheets orstrips, as pieces cut into any of a variety of shapes and/or sizes, orin a particulate form. The same concept applies to acellular tissuematrices produced from any of the above-listed tissues and organs.

Methods of Making and Using Acellular Tissue Matrices

The acellular tissue matrices of the invention can be produced by any ofa variety of methods. All that is required is that the steps used intheir production result in matrices with the above-described biologicaland structural properties. Particularly useful methods of productioninclude those described in U.S. Pat. Nos. 4,865,871 and 5,366,616 andco-pending U.S. application Ser. Nos. 09/762,174, 10/165,790, and10/273,780, the disclosures of which are incorporated herein byreference in their entirety.

In brief, the steps involved in the production of an acellular tissuematrix generally include harvesting the tissue from a donor (e.g., a pigor any of the species listed below), optionally slicing tissue in thehorizontal plane to generate multiple sheets, removal of the epithelialbasement membrane, chemical treatment so as to stabilize the tissue andavoid biochemical and structural degradation, together with or followedby cell removal under conditions which similarly preserve biological andstructural function. After thorough removal of dead and/or lysed cellcomponents below levels that may cause inflammation as well anybio-incompatible cell-removal agents, the acellular tissue matrix is inprinciple ready for implantation and only need be processed into adesired shape or size. Alternatively, the acellular tissue matrix can betreated with a cryopreservation agent and cryopreserved and, optionally,freeze dried, again under conditions necessary to maintain the describedbiological and structural properties of the matrix; after freeze drying,the tissue can be pulverized or micronized to produce a particulateacellular tissue matrix under similar function-preserving conditions.Alternatively, the acellular tissue matrix can be preserved by replacingmost of the water in the tissue with an agent such as glycerol such thatthe acellular tissue matrix contains, for example, approximately 85% byweight glycerol; the acellular tissue matrix can be stored in this formfor extended period at less than 20° C. All steps are generally carriedout under aseptic, preferably sterile, conditions.

The initial stabilizing solution arrests and prevents osmotic, hypoxic,autolytic, and proteolytic degradation, protects against microbialcontamination, and reduces mechanical damage that can occur with tissuesthat contain, for example, smooth muscle components (e.g., bloodvessels). The stabilizing solution generally contains an appropriatebuffer, one or more antioxidants, one or more oncotic agents, one ormore antibiotics, one or more protease inhibitors, and in some casesdepending on the tissue, a smooth muscle relaxant.

The tissue can then be placed in a processing solution to remove viablecells (e.g., epithelial cells, endothelial cells, smooth muscle cells,and fibroblasts) from the structural matrix without damaging thebiological and structural integrity of the collagen matrix. Theprocessing solution generally contains an appropriate buffer, salt, anantibiotic, one or more detergents, one or more agents to preventcross-linking, one or more protease inhibitors, and/or one or moreenzymes. Treatment of the tissue must be (a) with a processing solutioncontaining active agents at a concentration and (b) for a time periodsuch that the structural integrity of the matrix is maintained.

Where the tissue contains an epithelium (e.g., the epidermis of skin),the initial step in removing cells, prior to placement in the processingsolution(s) described above, can involve physical separation of theepithelium from the rest of the tissue using methods known in the art(see, e.g., Example 1). After removal of the epithelium, all, orsubstantially all, the epithelial basement membrane is removed anddiscarded. This is achieved, for example, by mechanical cutting.Appropriate methods are familiar to those in the art and can be, forexample, adaptations of methods used in the leather and tanningindustries.

After the tissue is decellularized, the resulting acellular tissuematrix can be incubated in a cryopreservation solution. This solutiongenerally contains one or more cryoprotectants (e.g., dimethyl sulfoxideor glycerol) to minimize ice crystal damage to the structural matrixthat can occur during freezing. If the acellular tissue matrix is to befreeze dried, the solution will generally also contain one or moredry-protective components, to minimize structural damage during dryingand may include a combination of an organic solvent and water whichundergoes neither expansion or contraction during freezing. As analternate method, the acellular tissue matrix can be fixed with acrosslinking agent such as glutaraldehyde and stored prior totransplantation. The cryoprotective and dry-protective agents can be thesame one or more substances. If the acellular tissue matrix is not goingto be freeze dried, it can be frozen by placing it (in a sterilizedcontainer) in a freezer at about −80° C., or by plunging it into sterileliquid nitrogen, and then storing at a temperature below −160° C. untiluse. The sample can be thawed prior to use by, for example, immersing asterile non-permeable vessel (see below) containing in a water bath atabout 37° C. or by allowing the acellular tissue matrix to come to roomtemperature under ambient conditions.

If the acellular tissue matrix is to be frozen and freeze dried,following incubation in the cryopreservation solution, it can bepackaged inside a sterile vessel that is permeable to water vapor yetimpermeable to bacteria, e.g., a water vapor permeable pouch or glassvial. One side of a preferred pouch consists of medical grade porousTyvek® membrane, a trademarked product of DuPont Company of Wilmington,Del. This membrane is porous to water vapor and impervious to bacteriaand dust. The Tyvek membrane is heat sealed to an impermeablepolyethylene laminate sheet, leaving one side open, thus forming atwo-sided pouch. The open pouch is sterilized by irradiation (e.g.,gamma irradiation) prior to use. The acellular tissue matrix isaseptically placed (through the open side) into the sterile pouch. Theopen side is then aseptically heat-sealed to close the pouch. Thepackaged acellular tissue matrix is thenceforth protected from microbialcontamination throughout subsequent processing steps.

The vessel containing the acellular tissue matrix is cooled to a lowtemperature at a specified rate which is compatible with the specificcryoprotectant to minimize the development of damaging hexagonal ice.See U.S. Pat. No. 5,336,616 for examples of appropriate coolingprotocols. The tissue is then dried at a low temperature under vacuumconditions, such that water vapor is removed sequentially without icerecrystallization.

At the completion of the drying of the samples in the water vaporpermeable vessel, the vacuum of the freeze drying apparatus is reversedwith a dry inert gas such as nitrogen, helium or argon. While beingmaintained in the same gaseous environment, the semipermeable vessel isplaced inside an impervious (i.e., impermeable to water vapor as well asmicroorganisms) vessel (e.g., a pouch), which is further sealed, e.g.,by heat and/or pressure. Where the acellular tissue matrix is frozen anddried in a glass vial, the vial is sealed under vacuum with anappropriate inert stopper and the vacuum of the drying apparatusreversed with an inert gas prior to unloading. In either case, the finalproduct is hermetically sealed in an inert gaseous atmosphere.

The freeze dried acellular tissue matrix can be stored under theseconditions for extended time periods under ambient refrigeratedconditions. Transportation may be accomplished via standard carriers andunder standard conditions relative to normal temperature exposure anddelivery times.

Generally (but not necessarily) the dried acellular tissue matrix isrehydrated prior to transplantation. It is important to minimize osmoticforces and surface tension effects during rehydration. The aim inrehydration is to augment the selective preservation of theextracellular support matrix. Appropriate rehydration can beaccomplished by, for example, an initial incubation of the driedacellular tissue matrix in an environment of about 100% relativehumidity, followed by immersion in a suitable rehydration solution.Alternatively, the dried tissue may be directly immersed in therehydration solution without prior incubation in a high humidityenvironment. Rehydration should not cause osmotic damage to the sample.Vapor rehydration should ideally achieve a residual moisture level of atleast 15% and fluid rehydration should result in a tissue moisture levelof between 20% and 70%. Depending on the particular acellular tissuematrix to be rehydrated, the rehydration solution can be, for example,normal saline, Ringer's lactate, or a standard cell culture medium.Where the acellular tissue matrix is subject to the action of endogenousproteases, collagenases, elastases or residual autolytic activity frompreviously removed cells, additives to the rehydration solution are madeand include protease inhibitors. Where residual free radical activity ispresent, agents to protect against free radicals are used includingantioxidants, and enzymatic agents that protect against free radicaldamage. Antibiotics may also be included to inhibit bacterialcontamination. Oncotic agents being in the form of proteoglycans,hyaluronan, dextran and/or amino acids may also be included to preventosmotic damage to the matrix during rehydration. Rehydration of a drysample is especially suited to this process as it allows rapid anduniform distribution of the components of the rehydration solution. Inaddition, the rehydration solutions may contain specific components notused previously, for example, diphosphonates to inhibit alkalinephosphatase and prevent subsequent calcification. Agents may also beincluded in the rehydration solution to stimulate neovascularization andhost cell infiltration following transplantation of the rehydratedacellular tissue matrix e.g., growth-factors, cytokines, chemoattractiveagents, cell-stimulatory factors, or genes.

Histocompatible, viable cells can be restored to the acellular tissuematrix to produce a permanently accepted graft that may be modeled bythe host. This is generally done just prior to, or after, placing of theacellular tissue matrix in a mammalian subject. Where the acellulartissue matrix has been freeze dried, it will be done after rehydration.In a preferred embodiment, histocompatible viable cells may be added tothe matrices by standard in vitro cell co-culturing techniques prior totransplantation, or by in vivo repopulation following transplantation.

The cell types used for reconstitution will depend on the nature of thetissue or organ to, or into, which the acellular tissue matrix isplaced. Cells with which the matrices can be repopulated include, butare not limited to, fibroblasts, embryonic stem cells (ESC), adult orembryonic mesenchymal stem cells (MSC), prochondroblasts, chondroblasts,chondrocytes, pro-osteoblasts, osteocytes, osteoclasts, monocytes,pro-cardiomyoblasts, pericytes, cardiomyoblasts, cardiomyocytes,gingival epithelial cells, or periodontal ligament stem cells.Naturally, the acellular tissue matrices can be repopulated withcombinations of two or more (e.g., two, three, four, five, six, seven,eight, nine, or ten) of these cell-types.

Following removal of cells, following freezing, following drying,following drying and rehydration, or following reconstitution of theacellular tissue matrix (whether or not frozen or dried) withappropriate cells, the acellular tissue matrix can be transported to theappropriate hospital or treatment facility. The choice of the finalcomposition of the product will be dependent on the specific intendedclinical application.

Reagents and methods for carrying out all the above steps are known inthe art. Suitable reagents and methods are described in, for example,U.S. Pat. No. 5,336,616.

Particulate acellular tissue matrices can be made from any of the abovedescribed non-particulate acellular tissue matrices by any process thatresults in the preservation of the biological and structural functionsdescribed above and, in particular, damage to collagen fibers, includingsheared fiber ends, should be minimized. Many known wet and dryingprocesses for making particulate matrices do not so preserve thestructural integrity of collagen fibers.

One appropriate method is described in co-pending U.S. patentapplication Ser. No. 09/762,174. The process is briefly described belowwith respect to a freeze dried dermal acellular tissue matrix but one ofskill in the art could readily adapt the method for use with freezedried acellular tissue matrices derived from any of the other tissueslisted herein.

The acellular dermal matrix can be cut into strips (using, for example,a Zimmer mesher fitted with a non-interrupting “continuous” cuttingwheel). The resulting long strips are then cut into lengths of about 1cm to about 2 cm. A homogenizer and sterilized homogenizer probe (e.g.,a LabTeck Macro homogenizer available from OMNI International,Warrenton, Va.) is assembled and cooled to cryogenic temperatures (i.e.,about −196° C. to about −160° C.) using sterile liquid nitrogen which ispoured into the homogenizer tower. Once the homogenizer has reached acryogenic temperature, cut pieces of acellular tissue matrix are addedto the homogenizing tower containing the liquid nitrogen. Thehomogenizer is then activated so as to cryogenically fracture the piecesof acellular tissue matrix. The time and duration of the cryogenicfracturing step will depend upon the homogenizer utilized, the size ofthe homogenizing chamber, and the speed and time at which thehomogenizer is operated, and are readily determinable by one skilled inthe art. As an alternative, the cryofracturing process can be conductedin a cryomill cooled to a cryogenic temperature.

The cryofractured particulate acellular tissue matrix is, optionally,sorted by particle size by washing the product of the homogenizationwith sterile liquid nitrogen through a series of metal screens that havealso been cooled to a cryogenic temperature. It is generally useful toeliminate large undesired particles with a screen with a relativelylarge pore size before proceeding to one (or more screens) with asmaller pore size. Once isolated, the particles can be freeze dried toensure that any residual moisture that may have been absorbed during theprocedure is removed. The final product is a powder (usually white oroff-white) generally having a particle size of about 1 micron to about900 microns, about 30 microns to about 750 microns, or about 150 toabout 300 microns. The material is readily rehydrated by suspension innormal saline or any other suitable rehydrating agent known in the art.It may also be suspended in any suitable carriers known in the art (see,for example, U.S. Pat. No. 5,284,655 incorporated herein by reference inits entirety). If suspended at a high concentration (e.g., at about 600mg/ml), the particulate acellular tissue matrices can form a “putty”,and if suspended at a somewhat lower concentration (e.g., about 330mg/ml), it can form a “paste”. Such putties and pastes can convenientlybe packed into, for example, holes, gaps, or spaces of any shape intissues and organs so as to substantially fill such holes, gaps, orspaces.

One highly suitable freeze dried acellular tissue matrix is producedfrom human dermis by the LifeCell Corporation (Branchburg, N.J.) andmarketed in the form of small sheets as AlloDerm®. Such sheets aremarketed by the LifeCell Corporation as rectangular sheets with thedimensions of, for example, 1 cm×2 cm, 3 cm×7 cm, 4 cm×8 cm, 5 cm×10 cm,4 cm×12 cm, and 6 cm×12 cm. The cryoprotectant used for freezing anddrying Alloderm is a solution of 35% maltodextrin and 10 mMethylenediaminetetraacetate (EDTA). Thus, the final dried productcontains about 60% by weight acellular tissue matrix and about 40% byweight maltodextrin. The LifeCell Corporation also makes an analogousproduct from pig dermis as XenoDerm™ having the same proportions ofacellular tissue matrix and maltodextrin as AlloDerm. In addition, theLifeCell Corporation markets a particulate acellular dermal matrix madeby cryofracturing AlloDerm (as described above) under the name Cymetra®.The particle size for Cymetra is in the range of about 60 microns toabout 150 microns as determined by mass.

The particles of particulate or pulverized (powdered) acellular tissuematrices of the invention will be less than 1.0 mm in their longestdimension. Pieces of acellular tissue matrix with dimensions greaterthan this are non-particulate acellular matrices.

The form of acellular tissue matrix used in any particular instance willdepend on the tissue or organ to which it is to be applied. Generallynon-particulate acellular tissue matrices that are provided in dry form(e.g., AlloDerm) are rehydrated in a sterile physiological solution(e.g., saline) before use. However they can also be used dry.

Sheets of acellular tissue matrix (optionally cut to an appropriatesize) can be: (a) wrapped around a tissue or organ that is damaged orthat contains a defect; (b) placed on the surface of a tissue or organthat is damaged or has a defect; or (c) inserted into a cavity, gap, orspace in the tissue or organ. Such cavities, gaps, or spaces can be, forexample: (i) of traumatic origin (e.g. an incisional hernia or aninfection-related defect), (ii) due to removal of diseased tissue (e.g.,infarcted myocardial tissue), or (iii) due to removal of malignant ornon-malignant tumors. The acellular tissue matrices can be used toaugment or ameliorate underdeveloped tissues or organs or to augment orreconfigure deformed tissues or organs. One or more (e.g., one, two,three, four, five, six, seven, eight, nine, ten, 12, 14, 16, 18, 20, 25,30, or more) such strips can be used at any particular site. The graftscan be held in place by, for example, sutures, staples, tacks, or tissueglues or sealants known in the art. Alternatively, if, for example,packed sufficiently tightly into a defect or cavity, they may need nosecuring device. Particulate acellular tissue matrices can be suspendedin a sterile pharmaceutically acceptable carrier (e.g., normal saline)and injected via hypodermic needle into a site of interest.Alternatively, the dry powdered matrix or a suspension can be sprayedinto or onto a site or interest. A suspension can be also be poured intoor onto a particular site. In addition, by mixing the particulateacellular tissue matrix with a relatively small amount of liquidcarrier, a “putty” can be made. Such a putty, or even dry particulateacellular tissue matrix, can be layered, packed, or encased in any ofthe gaps, cavities, or spaces in organs or tissues mentioned above.Moreover, a non-particulate acellular tissue matrix can be used incombination with particulate acellular tissue matrix. For example, acavity in bone could be packed with a putty (as described above) andcovered with a sheet of acellular tissue matrix.

It is understood that an acellular tissue matrix can be applied to atissue or organ in order to repair or regenerate that tissue or organand/or a neighboring tissue or organ. For example, a strip or multiplestrips of acellular tissue matrix can be used to augment primary ventralhernia repairs, or for larger defects can be used to substitute formissing or excised fascia and muscle, thus providing protection andmechanical support in abdominal wall defects. A strip of acellulartissue matrix can be wrapped around a critical gap defect of a long boneto generate a periosteum equivalent surrounding the gap defect and theperiosteum equivalent can in turn stimulate the production of bonewithin the gap in the bone. Similarly, by implanting an acellular tissuematrix in a dental extraction socket, injured gum tissue can be repairedand/or replaced and the “new” gum tissue can assist in the repair and/orregeneration of any bone in the base of the socket that may have beenlost as a result, for example, of tooth extraction. In regard to gumtissue (gingiva), receding gums can also be replaced by injection of asuspension, or by packing of a putty of particulate acellular tissuematrix into the appropriate gum tissue. Again, in addition to repairingthe gingival tissue, this treatment can result in regeneration of bonelost as a result of periodontal disease and/or tooth extraction.Compositions used to treat any of the above gingival defects can containone or more other components listed herein, e.g., demineralized bonepowder, growth factors, or stem cells.

Both non-particulate and particulate acellular tissue matrices can beused in combination with other scaffold or physical support components.For example, one or more sheets of acellular tissue matrix can belayered with one or more sheets made from a biological material otherthan acellular tissue matrix, e.g., irradiated cartilage supplied by atissue bank such as LifeNet, Virginia Beach, Va., or bone wedges andshapes supplied by, for example, the Osteotech Corporation, Edentown,N.J. Alternatively, such non-acellular tissue matrix sheets can be madefrom synthetic materials, e.g., polyhydroxy-alkanoates, such as thatsupplied by Tepha, Inc., Boston, Mass. Other suitable scaffold orphysical support materials are disclosed in U.S. Pat. No. 5,885,829, thedisclosure of which is incorporated herein by reference in its entirety.It is understood that such additional scaffold or physical supportcomponents can be in any convenient size or shape, e.g., sheets, cubes,rectangles, discs, spheres, or particles (as described above forparticulate acellular tissue matrices).

Other active substances that can be mixed with particulate acellulartissue matrices or impregnated into non-particulate acellular tissuematrices include bone powder, demineralized bone powder, and any ofthose disclosed above.

Factors that can be incorporated into the acellular tissue matrices,administered to the placement site of an acellular tissue matrix graft,or administered systemically include any of a wide range of cell growthfactors, angiogenic factors, differentiation factors, cytokines,hormones, and chemokines known in the art. Any combination of two ormore of the factors can be administered to a subject by any of the meansrecited below. Examples of relevant factors include fibroblast growthfactors (FGF) (e.g., FGF1-10), epidermal growth factor, keratinocytegrowth factor, vascular endothelial cell growth factors (VEGF) (e.g.,VEGF A, B, C, D, and E), platelet-derived growth factor (PDGF),interferons (IFN) (e.g., IFN-α, β, or γ), transforming growth factors(TGF) (e.g., TGFα or β), tumor necrosis factor-α, an interleukin (IL)(e.g., IL-1-IL-18), Osterix, Hedgehogs (e.g., sonic or desert), SOX9,bone morphogenic proteins, parathyroid hormone, calcitoninprostaglandins, or ascorbic acid.

Factors that are proteins can also be delivered to a recipient subjectby administering to the subject: (a) expression vectors (e.g., plasmidsor viral vectors) containing nucleic acid sequences encoding any one ormore of the above factors that are proteins; or (b) cells that have beentransfected or transduced (stably or transiently) with such expressionvectors. Such transfected or transduced cells will preferably be derivedfrom, or histocompatible with, the recipient. However, it is possiblethat only short exposure to the factor is required and thushisto-incompatible cells can also be used. The cells can be incorporatedinto the acellular tissue matrices (particulate or non-particulate)prior to the matrices being placed in the subject. Alternatively, theycan be injected into an acellular tissue matrix already in place in asubject, into a region close to an acellular tissue matrix already inplace in a subject, or systemically. Naturally, administration of theacellular tissue matrices and/or any of the other substances or factorsmentioned above can be single, or multiple (e.g., two, three, four,five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40, 50, 60, 80,90, 100, or as many as needed). Where multiple, the administrations canbe at time intervals readily determinable by one skilled in art. Dosesof the various substances and factors will vary greatly according to thespecies, age, weight, size, and sex of the subject and are also readilydeterminable by a skilled artisan.

Conditions for which the matrices can be used are multiple. Thus, forexample, they can be used for the repair of bones and/or cartilage withany of the above-described damage or defects. Both particulate andnon-particulate acellular tissue matrices can be used in any of theforms and by any of the processes listed above. Bones to which suchmethods of treatment can be applied include, without limitation, longbones (e.g., tibia, femur, humerus, radius, ulna, or fibula), bones ofthe hand and foot (e.g., calcaneas bone or scaphoid bone), bones of thehead and neck (e.g., temporal bone, parietal bone, frontal bone,maxilla, mandible), or vertebrae. As mentioned above, critical gapdefects of bone can be treated with acellular tissue matrices. In suchcritical gap defects, the gaps can be filled with, example, a putty ofparticulate acellular tissue matrix or packed sheets of acellular tissuematrix and wrapped with sheets of acellular tissue matrix.Alternatively, the gaps can be wrapped with a sheet of acellular tissuematrix and filled with other materials (see below). In all these boneand/or cartilage treatments, additional materials can be used to furtherassist in the repair process. For example, the gap can be filledcancellous bone and or calcium sulfate pellets and particulate acellulartissue matrices can be delivered to sites of bone damage or bone defectsmixed with demineralized bone powder. In addition, acellular tissuematrices can be combined with bone marrow and/or bone chips from therecipient.

Acellular tissue matrices can also be used to repair fascia, e.g.,abdominal wall fascia or pelvic floor fascia. In such methods, strips ofacellular tissue matrix are generally attached to the abdominal orpelvic floor by, for example, suturing either to the surrounding fasciaor host tissue or to stable ligaments or tendons.

Infarcted myocardium is another candidate for remodeling repair byacellular tissue matrices. Contrary to prior dogma, it is now known thatnot all cardiac myocytes have lost proliferative and thus regenerativepotential [e.g., Beltrami et al. (2001) New. Engl. J. Med.344:1750-1757; Kajstura et al. (1998) Proc. Nat'l. Acad. Sci. USA95:8801-8805]. Moreover, stem cells, present for example in bone marrowand blood and as pericytes associated with blood vessels, candifferentiate to cardiac myocytes. Either the infarcted tissue itselfcan be removed and replaced with a sheet of acellular tissue matrix cutto an appropriate size or a suspension of particulate acellular tissuematrix can be injected into the infarcted tissue. Congenital hearthypoplasia, or other structural defects, can be repaired by, forexample, making an incision in the tissue, expanding the gap created bythe incision, and inserting a sheet of acellular tissue matrix cut tothe desired size, or placing sheets of acellular tissue matrix on theepicardial and endocardial surfaces and placing particulate acellulartissue matrix between them. It is understood that, in certainconditions, creating a gap by incision may not be sufficient and it maybe necessary to excise some tissue. Naturally, one of skill in the artwill appreciate that the acellular tissue matrices can be used similarlyto repair damage to or defects in other types of muscle, e.g., ureter orbladder or skeletal muscle such as biceps, pectoralis, or latissimus.

Moreover, sheets of acellular tissue matrix can be used to repair orreplace damaged or removed intestinal tissue, including the esophagus,stomach and small and large intestines. In this case, the sheets ofacellular tissue matrix can be used to repair perforations or holes inthe intestine. Alternatively, a sheet of acellular tissue matrix can beformed, for example, into a cylinder, which can be used to fill a gap inthe intestine (e.g., a gap created by surgery to remove a tumor or adiseased segment of intestine). Such methods can be used to treat, forexample, diaphragmatic hernias. It will be understood that an acellulartissue matrix in sheet form can also be used to repair the diaphragmitself in this condition as well as in other conditions of the diaphragmrequiring repair or replacement, or addition of tissue.

Donors of Tissue for Making Acellular Tissue Matrices and Recipients ofAcellular Tissue Matrices

The acellular tissue matrices of the invention are generally made fromone or more individuals of a species other than that of the recipient ofthe acellular tissue matrix graft. Thus, for example, an acellulartissue matrix can be made from a pig and be implanted in a humanpatient. In addition, while recipients of GTδ acellular tissue matriceswill generally be of a GT− species, this also is not absolutelyrequired. Since GT+ animals express αgal epitopes they would generallybe expected not to produce antibody specific for αgal epitopes. Thus, insuch a recipient it is not relevant whether an acellular tissue matrixto be placed in the recipient contains αgal epitopes or not. Speciesthat can serve as recipients of acellular tissue matrices thus include,without limitation, humans, non-human primates (e.g., monkeys, baboons,or chimpanzees), pigs, cows, horses, goats, sheep, dogs, cats, rabbits,guinea pigs, gerbils, hamsters, rats, or mice. Preferred recipients areof GT− species, e.g., humans and Old World primates (e.g. African greenmonkeys, cynamolgus monkeys, macaques, Vervet monkeys, and baboons).Species from which animals genetically modified so as to lack, orsubstantially lack, αgal epitopes can be made as a source of tissues forthe production of the acellular tissue matrices of the invention includeall of the above except the GT− species, i.e., human and Old Worldprimates. A preferred genetic modification is the disruption of bothalleles of the αGT gene in all the cells of such animals. Otherappropriate genetically modified animals include, without limitation,α(1,2)fucosyltransferase or α(2,6)sialyltransferase transgenic animals[see, for example, U.S. Pat. No. 6,166,288; and Sharma et al. (1996)Proc. Natl. Acad. Sci. USA 93(14):7190-7195, the disclosures of whichare incorporated herein by reference in their entirety].

Methods of making transgenic animals, and in particular gene-disruptedtransgenic animals, are well known in the art. Indeed, both GTδ mice andGTδ pigs have been generated [see, for example: U.S. Pat. No. 5,849,991;Lai et al. (2002) Science 295(5557):1089-1092; Dai et al. (2002) Nat.Biotechnol. 20(3):251-255; Phelps et al. (2003) Science299(5605):411-414; and Lai et al. (2003) Cloning Stem Cells 5(4):233-241, the disclosures of all of which are incorporated herein byreference in their entirety].

Methods of making gene-disrupted animals involve incorporating into thegermline of an individual of a species a disrupted form of a gene ofinterest. The gene can be disrupted so that no protein product (e.g.,αGT) is produced or a protein product is produced that lacks theactivity, or substantially lacks the activity, of the protein. As usedherein, a αGT protein “substantially lacking αGT activity” is an αGTprotein that has less than 5% (e.g., less than: 4%; 2%; 1%; 0.1%; 0.01%;0.001%; or even less than 0.001%) of the ability of wild-type αGT togenerate αgal epitopes. Methods of disrupting genes, and in particular,the αGT gene, are known in the art (see above) and generally involve theprocess known as homologous recombination. In this process, one or bothcopies of a wild-type gene of interest in an appropriate target cell(see below) is/are disrupted by inserting, using an appropriate geneticconstruct, a sequence into the wild-type gene(s) such that no transcriptis produced from the gene(s), a transcript is produced from which noprotein is translated, or a transcript is produced from which a proteinis produced that lacks, or substantially lacks, the activity of theprotein of interest. In such constructs, there is generally all or partof the genomic sequence of the gene of interest and, within that genomicsequence, a sequence that will disrupt expression of the gene in one ofthe ways described above. The sequence used to disrupt expression of thegene will frequently be a sequence encoding a protein that endowsantibiotic resistance (e.g., neomycin resistance) on target cells thathave incorporated the construct into their genomes. Such a codingsequence facilitates the in vitro selection of cells that haveincorporated the genetic construct into their genomes. Additional drugselection methodologies known in the art can be used to select cells inwhich recombination between the construct and at least one copy of thetargeted gene has occurred.

In earlier methods of generating gene disrupted animals, totipotentcells (i.e., cells capable of giving rise to all cell types of anembryo) were used as target cells. Such cells included, for exampleembryonic stem (ES) cells (in the form of ES cell lines) or fertilizedeggs (oocytes). A population of ES cells in which at least one copy ofthe gene of interest is disrupted is injected into appropriateblastocysts and the injected blastocysts are implanted into fostermothers. Alternatively, fertilized eggs injected with thegene-disrupting construct of interest are implanted in the fostermothers. Moreover, oocytes implanted in foster mothers can be those thathave been enucleated and injected with nuclei from successfullygene-disrupted ES cells [Campbell et al. (1996) Nature 380: 64-66].Resulting mutation-containing offspring arising in such mother fostermothers are identified and, from these founder animals, distinct animallines are produced using breeding and selection methods known to thosein the art.

More recently methods for making standard and gene-disrupted transgenicanimals have employed somatic cells (e.g., fetal fibroblasts) as targetcells for the gene-disruption (see below). Since such cells grow muchfaster and are more easily handled in vitro than, for example, ES cells,the gene disruption and subsequent gene-disrupted cell selectionprocedures are greatly facilitated using them. Having selected a line ofsuccessfully gene-disrupted somatic cells in vitro, using any of anumber of procedures (e.g., cell fusion or nuclear transplantation),nuclei from the successfully gene-disrupted somatic cells areincorporated into totipotent cells (e.g., ES cells or oocytes), whichare then handled as described above for the original methods.

Most commonly, the gene disruption procedures result in disruption ofonly one allele of a gene of interest. In these cases, animals bred asdescribed to have the gene disruption in all their somatic and germcells, will have only one allele disrupted, i.e., the animals will beheterozygous for the disrupted gene. Breeding of such a heterozygotesand appropriate selection procedures familiar to artisans in the fieldcan then be used to derive animals that are homozygous for the disruptedgene. Naturally, such breeding procedures are not necessary where thegene disruption procedure described above resulted in disruption of bothalleles of the gene of interest.

The above-described procedures have also been used to generatedisruptions of genes other than the GT gene in species such as, withoutlimitation, mice, sheep, and pigs Furthermore, in light of the fact thatsomatic cell procedures (as described above) have been successfully usedto generate GTδ pigs as well as standard non-gene disrupted transgenicsheep and cattle [Wilmut et al. (1997) Nature 385:81-813; Kato et al.(1998) Science 282:2095-2098; Schnieke et al. (1997) Science 278:2130-2133; Cibelli et al. (1998) Science 280: 1256-1258], the somaticcell-based procedures can readily be adapted for the generation ofgene-disrupted (including GTδ) animals of a wide variety of species.

The following examples serve to illustrate, not limit, the invention.

EXAMPLES Example 1 Materials and Methods

Preparation of Matrices Used for Implantation Experiments

For the experiments described in Examples 2-4 below, acellular dermalmatrices (ADM) from human donors, wild type pigs, and GTδ pigs wereproduced using LifeCell's proprietary methodology. The methodology formaking ADM from human donors (Alloderm) is described in detail below;the process used for making ADM from pig skin is essentially identicalexcept as otherwise stated.

Human donor skin was obtained from various U.S. tissue banks andhospitals throughout the nation that collected skin samples usingstandard procedures from deceased donors after obtaining the consentfrom family members. Procured skin was placed in RPMI 1640 tissueculture media containing antibiotics (penicillin and streptomycin) andwas shipped to LifeCell's facility in Branchburg, N.J., on wet ice, inthe same media. On arrival, the temperature of the skin tissue containerwas measured and the skin tissue was discarded if the temperature wasabove 10° C. The RPMI 1640 media was changed under aseptic condition andthe skin was stored at 4° C., while serological tests (e.g., forsyphilis, HIV I and II, hepatitis B surface antigen, hepatitis C virusand HTLV I and II) were performed. The skin was then transferred to apre-freezing aqueous solution of 35% w/v maltodextrin. After 2 to 4hours, the skin was frozen and stored in a −80° C. freezer until it wasprocessed as described below.

Frozen skin was thawed at 37° C. in a water bath until no visible icewas left. The pre-freezing solution was drained before furtherprocessing, consisting of the following steps: (i) de-epidermization;(ii) de-cellularization; (iii) wash.

(i) De-epidermization: Skin epidermis was removed by incubating thetissue sample with gentle agitation in a de-epidermizing solution (1MNaCl, 0.5% w/v Triton X100, 10 mM EDTA) for 8-32 hours for human skinand 30-60 hour for porcine skin at room temperature. The epidermal layerwas removed from dermis. The epidermis was discarded and the dermisretained for further processing.

(ii) De-cellularization: In order to remove cellular components, thedermis was rinsed for 5 to 60 minutes with a de-cellularizing solution(2% w/v sodium deoxycholate, 10 mM EDTA, 10 mM HEPES buffer, pH7.8-8.2), and then incubated with gentle agitation in that solution for12-30 hours at room temperature.

(iii) Wash: The washing regimen serves to wash out dead cells, celldebris, and residual chemicals used in the previous processing steps.The de-cellularized dermis was transferred to a first wash solution(phosphate buffered saline (PBS) containing 0.5% w/v Triton X-100 and 10mM EDTA) which was then incubated with gentle agitation for 5 to 60minutes at room temperature. The dermis was then subjected to threesequential washes in a second wash solution (PBS containing 10 mM EDTA)with gentle agitation at room temperature. The first two washes wereshort (15-60 minutes each) and the third wash was long (6-30 hours).

After the wash regimen, the resulting ADM were freeze-dried usingstandard methods. Immediately prior to implantation, the samples wererehydrated in normal saline, cut into appropriate sizes (about 1 cm²),and then used for the experiments described in Examples 2-4.

Operative Procedure

Old World Primates (African green monkeys (Ceropithecus aethiops); 5-7year-old males) were implanted with ADM made from either wild type pigs(XenoDerm™), human donors (Alloderm®), or GT5 pigs [Lai, L., et al.(2002)] (GalDerm™). The ADM were placed into four subdermal pocketscreated in their upper backs.

The ADM, which had been freeze dried, were prehydrated in excess salinefor about 30 minutes. Each monkey was implanted with 1 piece of ADM persite (each piece being approximately 1 cm²). Thus each animal receivedfour implants of the same ADM, the four implants corresponding to fourtime points, i.e., day 0, day-7, day-21, and day-35. Three animals wereimplanted with each ADM (n=3). Implants with associated adherent hosttissue were removed at each time point by excision of the samplematerial. Explants were bisected with a scalpel; half was placed informalin fixative (and stored at 4° C.), and the other half was placedin sucrose cryoprotectant (and stored at 4° C.) for histologicalanalysis.

Detection of αgal Epitopes

Detection of αgal epitopes in the ADM was done by directimmunohistology. AlloDerm, XenoDerm, and GalDerm, were immunostainedwith a monoclonal mouse IgG3 anti-αgal antibody. Antibody binding wasdetected using a horseradish peroxidase (HRP)-conjugated goat anti-mouseIgG3 secondary antibody, and visualized with 3,3′-diaminobenzidine(DAB).

Histological Analysis

Histological analysis of the implanted material was done by standardhematoxylin and eosin (H&E) staining of formalin-fixed sections of eachexplant.

Serum Analysis

The occurrence of a humoral immune response to the implant was assessedby determining the titer of implant-specific antibody in the sera of theanimals at various times after implantation. Blood was drawn at timepoints throughout the study, i.e., on day-0, day-7, day-21, and day-35.Serum was isolated and stored at −70° C. prior to analysis.

Serum dilutions were tested for relative level of antibody specific forantigens of implanted tissue via a modified sandwich ELISA procedure.Antibody titration curves were derived from serum samples obtained ondays 7, 21, and 35 compared and curves were compared with valuesobtained with day-0 (pre-implant) serum samples.

ELISA plates were prepared as follows. Samples of the individual ADMused for implantation were freeze-dried and then powdered bycryofracture using a Spexfreezer™ mill (Spex Industries, Edison, N.J.).Each sample of powdered ADM was suspended in phosphate buffered saline(PBS) at a final concentration of 0.5 mg/mL. Aliquots (of 50 μL) of thesuspensions were added to the wells of 96-well immulon ELISA plates andallowed to air dry. The powdered ADM were fixed to the well bottoms byadding 0.2% glutaraldehyde to the wells and incubating the plates for 30minutes at room temperature. The plates were then washed and the wellcontents blocked with 0.2 M glycine. After further washing, bovine serumalbumin (BSA; 1% in Tris-buffered saline (TBS)) was added to the wellsto block non-specific protein-binding sites. Test sera obtained from themonkeys at the indicated timepoints were diluted in 1% BSA. Afterdiscarding the BSA solution used to block non-specific protein-bindingsites, aliquots (50 μL) of serum dilutions were placed in the microtiterwells and incubated for 2 hours at room temperature. The plates werewashed three times with TBS containing 0.1% Tween. After washing of theplates to remove unbound material in the added serum dilutions, anoptimal dilution of goat anti-human IgG secondary antibody conjugated toalkaline phosphatase (Sigma, St. Louis, Mo.) was added to each well ofthe plate. The plates were incubated at room temperature for 1 hour andthen washed five times with TBS containing 0.1% Tween. The pNPP(4-nitrophenyl phosphate) substrate was added to each of the wells, andthe plates were incubated for 1 hour at room temperature to facilitate acolor reaction. The optical density at 405 nm (OD₄₀₅) of each well wasread using a microwell plate spectrophotometer. The OD₄₀₅ values wereused to generate titration curves.

Example 2 Presence and Absence of αgal Epitopes in ADM

To assess the presence of αgal epitopes in implant materials, AlloDerm,XenoDerm, and GalDerm (FIGS. 1A, 1B, and 1C, respectively) were stainedwith a mouse IgG3 monoclonal anti-αgal antibody, a HRP-conjugated goatanti-mouse IgG3 secondary antibody, and the HRP substrate DAB. Controlsto detect non-specific binding of the secondary antibody were included;these control samples were exposed to the secondary antibody only. Thecontrol for staining of GalDerm is shown in FIG. 1D. AlloDerm andXenoDerm samples were also counter-stained with hematoxylin. XenoDerm(FIG. 1B) showed diffuse staining with the anti-αgal antibody throughoutthe matrix coincident with edges of collagen bundles. On the other hand,no staining with the anti-αgal antibody was seen in AlloDerm (FIG. 1A).Similarly, GalDerm (FIG. 1C) showed no signal with the anti-αgalantibody above that obtained in the control sample (FIG. 1D). Theseresults demonstrated the absence of αgal epitopes on GalDerm andAlloDerm and their presence on XenoDerm.

Example 3 Histological Analysis of Explants

Histological evaluation of the implants 7 days after implantationrevealed cellular infiltrates in the peripheral regions of all threetypes of ADM. Histological evaluation of the implants at day 35 afterimplantation showed significant differences between the three types ofADM. AlloDerm (FIGS. 2A and 2B) and GalDerm (FIGS. 4A and 4B) showedrepopulation with fibroblast-like cells throughout the implants. On theother hand, in XenoDerm (FIGS. 3A, 3B and 3C) there were extensivecellular infiltrates in the peripheral region, these infiltrates beingcomposed primarily of inflammatory cells, and there was no evidence ofcellular repopulation of the interior of the implant.

Example 4 Analysis of Sera from Implant Recipients

The presence of a humoral (antibody) immune response against implantedmaterial in the test recipient animals was tested by measuring theproduction of tissue-specific antibodies in sera obtained from theseanimals. Antibody binding to the corresponding implant material wasmeasured by a modified ELISA assay (see Example 1). The production ofantibodies at various time points in serum samples was assessed bycomparison with pre-transplant values. It is clear that sera from allthe animals contained preformed ADM-binding antibodies, i.e.,ADM-binding antibodies were detected in sera obtained on day 0 from allanimals (FIG. 5). The serum dilution curves show clear increases in thetiters of anti-XenoDerm IgG antibodies in sera obtained 21 days afterimplantation of XenoDerm (FIG. 5A). The levels of these antibodiesremained elevated through the 35 day time point. In contrast, in monkeysimplanted with GalDerm (FIG. 5B) and AlloDerm (FIG. 5C) there was noelevation in the titers of IgG ADM-binding antibodies (at any timepoint) compared to pre-implant IgG levels.

To further characterize the specificity of the antibodies in sera fromXenoDerm-implanted animals that bound to XenoDerm in the ELISA, acompetition ELISA in the presence or absence of αgal was performed. Thebinding to XenoDerm of antibodies in sera obtained at all three timepoints from all three monkeys implanted with XenoDerm was completelyinhibited by 10 mM αgal (data not shown). These results indicate that:(a) antibodies in sera from XenoDerm-implanted animals that bound toXenoDerm were anti-αgal antibodies; and (b) in these animals there wasno significant XenoDerm-specific antibody response to antigens otherthan αgal epitopes.

The data indicate in toto that the transplantation of XenoDerm into αgaldeficient monkeys caused the monkeys to mount a systemic antibodyresponse and that this antibody response is directed largely, if notexclusively, against αgal epitopes in the XenoDerm. GalDerm and AlloDermimplants, on the other hand, initiated no such antibody response.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1. An acellular tissue matrix comprising: a decellularizedcollagen-containing dermal tissue of a pig genetically modified tocomprise a disruption of a α-1,3-galactosyltransferase gene such thattissues in the pig lack, or substantially lack, galactoseα-1,3-galactose epitopes, wherein the acellular tissue matrix lacks, orsubstantially lacks, epithelial basement membrane.
 2. The acellulartissue matrix of claim 1, wherein the tissue is selected from at leastone of fascia, pericardium, dura, umbilical cord, placenta, cardiacvalve, ligament, tendon, artery, vein, neural connective tissue,intestine, bladder, and ureter.
 3. The acellular tissue matrix of claim1, wherein the acellular tissue matrix is in non-particulate form. 4.The acellular tissue matrix of claim 1, wherein the acellular tissuematrix is in particulate form.
 5. The acellular tissue matrix of claim1, wherein the genetic modification comprises a disruption of bothalleles of an α-1,3 galactosyl transferase (αGT) gene.
 6. A method ofmaking an acellular tissue matrix, the method comprising: (a) providinga collagen-containing tissue from a pig genetically modified to comprisea disruption of a α-1,3-galactosyltransferase gene so that tissues inthe pig lack, or substantially lack, galactose α-1,3-galactose epitopes;and (b) processing the tissue so as to render the tissue acellular andlacking, or substantially lacking, an epithelial basement membrane, theprocessing resulting in the production of an acellular tissue matrix. 7.The method of claim 6, wherein the genetic modification comprises adisruption of both alleles of an α-1,3 galactosyl transferase (αGT)gene.
 8. The method of claim 6, further comprising freezing theacellular tissue matrix.
 9. The method of claim 6, further comprisingfreeze-drying the acellular tissue matrix.
 10. The method of claim 6,further comprising: (a) pulverizing the acellular tissue matrix; or (b)rendering the acellular tissue matrix particulate in form.
 11. Themethod of claim 6, wherein the process comprises removing and discardingepidermis.
 12. A method of treatment, the method comprising: (a)identifying a mammalian subject as having an or organ, or tissue, inneed of repair or amelioration; and (b) placing a composition comprisingthe acellular tissue matrix of claim 1 in or on the organ or tissue. 13.The method of claim 12, wherein the subject is a human.
 14. The methodof claim 12, wherein the acellular tissue matrix is in non-particulateform.
 15. The method of claim 12, wherein the acellular tissue matrix isin particulate form.
 16. The method of claim 12, wherein the geneticmodification comprises disruption of both alleles of an α-1,3 galactosyltransferase (αGT) gene.
 17. The method of claim 12, further comprisingadministration to the subject of at least one agent selected from a cellgrowth factor, an angiogenic factor, a differentiation factor, acytokine, a hormone, and a chemokine.
 18. The method of claim 17,wherein the at least one agent is in the composition placed in thesubject.
 19. The method of claim 17, wherein the administrationcomprises injecting or infusing the at least one agent into themammalian subject separately from the composition.
 20. The method ofclaim 12, wherein the organ or tissue of the subject is selected from atleast one of skin, bone, cartilage, meniscus, dermis, myocardium,periosteum, artery, vein, stomach, small intestine, large intestine,diaphragm, tendon, ligament, neural tissue, striated muscle, smoothmuscle, bladder, urethra, ureter, and gingiva.
 21. The method of claim12, wherein the organ or tissue of the subject is abdominal wall fascia.22. The method of claim 12, wherein the composition further comprisesdemineralized bone powder.
 23. The method of claim 20, wherein the organor tissue of the subject is gingiva, and wherein the gingiva is, or isproximal to, receding gingiva.
 24. The method of claim 20, wherein theorgan or tissue of the subject is gingiva, and wherein the gingivacomprises a dental extraction socket.